CRISPR/Cas12a Colorimetric Sensor for Pathogen Detection
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Solution Overview
Problem
Current methods for detecting pathogens, such as culture-based, immunoassay, and PCR systems, are either resource-intensive, prone to contamination, or require expensive equipment, making them unsuitable for rapid and inexpensive on-site detection, especially for airborne pathogens.
Innovation Solution
A dual enzyme amplification-based colorimetric sensor system utilizing a CRISPR/Cas12a complex, urease-conjugated single-stranded DNA, and a pH indicator, which activates a color change in the presence of target DNA, enabling visual detection using a smartphone application.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If culture-based methods are used for pathogen detection, then detection can be performed with simple equipment, but the method is time-consuming, resource-intensive, and susceptible to contamination
Solution Approach 1:
The patent replaces traditional mechanical/culture-based detection methods with an optical detection system using colorimetric changes. The CRISPR/Cas12a complex activates in the presence of target DNA, triggering a color change in the solution that can be detected visually or with a smartphone, eliminating the need for incubation periods and complex culture equipment while dramatically reducing detection time.
Solution Approach 2:
The patent introduces an intermediary colorimetric indicator system that mediates between the molecular recognition event (CRISPR/Cas12a binding to target DNA) and the observable detection result. The indicator converts the molecular interaction into a visible color change, allowing rapid detection without direct observation of microbial growth or complex instrumental analysis.
2Measurement precision
If immunoassay and PCR methods are used for pathogen detection, then detection sensitivity and specificity are improved, but expensive reagents and analytical equipment are required
Solution Approach 1:
The patent employs disposable, low-cost reagents including the CRISPR/Cas12a complex, guide RNA, and colorimetric indicators that can be prepared in simple buffers. These reagents replace expensive PCR kits and immunoassay reagents, enabling high-sensitivity detection without requiring expensive analytical equipment. The system can be performed with basic laboratory equipment or even a smartphone camera.
Solution Approach 2:
The patent substitutes complex analytical equipment (PCR machines, sequencers, flow cytometers) with a simple optical detection system based on colorimetric changes. The CRISPR/Cas12a-mediated color change can be observed with the unaided eye or captured by a smartphone camera, replacing millions-dollar instruments with accessible technology while maintaining high detection sensitivity.
3Measurement precision
If electrochemical technology-based methods are used for pathogen detection, then detection accuracy is improved, but uniformity is poor due to use of non-commercial electrodes
Solution Approach 1:
The patent replaces electrochemical detection methods with an optical colorimetric detection system. This substitution eliminates the reliability issues associated with non-commercial electrodes and variable electrochemical conditions. The colorimetric readout provides consistent, reproducible results that are independent of electrode manufacturing variations, while maintaining high detection accuracy through the specificity of CRISPR/Cas12a molecular recognition.
4Ease of manufacture
If plasmonic nanoparticles are used for colorimetric detection, then color change detection is enabled, but synthesis is complicated and signal reproducibility is poor
Solution Approach 1:
The patent replaces complex-to-synthesize plasmonic nanoparticles with simple, commercially available colorimetric indicators and enzymes. The system uses readily obtainable reagents that can be prepared in standard laboratory conditions, eliminating the need for specialized nanoparticle synthesis equipment and expertise. This approach maintains colorimetric detection capability while dramatically simplifying the manufacturing process and improving signal reproducibility.
Solution Approach 2:
The patent extracts the essential function of plasmonic nanoparticles (colorimetric signal generation) and replaces it with a simpler biochemical system based on enzyme-catalyzed color development. By taking out the nanoparticle component and substituting it with a biochemical colorimetric system, the patent achieves the same detection capability with simpler, more reproducible reagents that do not require specialized synthesis procedures.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This system allows for rapid, sensitive, and cost-effective detection of pathogens down to sub-ng levels without the need for specialized equipment, facilitating on-site identification of airborne pathogens.
Implementation Method 1
a CRISPR/Cas12a complex comprising a CRISPR/Cas12a protein, and a guide RNA including a region that binds to the CRISPR/Cas12a protein and a guide sequence that hybridizes to a target DNA; nanoparticles immobilized with urease-conjugated single-stranded DNA (ssDNA)
Implementation Method 2
confirming, when single-stranded DNA (ss-DNA) of the nanoparticles immobilized with the urease-conjugated ssDNA is cleaved by activation of the CRISPR/Cas12a complex, a color change of a solution by the cleaved urease
Implementation Method 3
a pH indicator
Data Source
AI summary
The present disclosure relates to a dual enzyme amplification-based colorimetric sensor system for on-site detection of pathogens. The colorimetric sensor system according to the present disclosure may comprise a combination of the CRISPR/Cas12a system with an enzymatic reaction of urease, thereby facilitating on-site detection of pathogens without separate analytical equipment by analyzing the color change through dual enzyme amplification. In addition, it is possible to selectively change the target by changing the crRNA sequence depending on the target pathogen to be detected, which has the advantage of being applicable to various types of pathogens without limitation. Further, the present disclosure can be used as a point of care service (PoC) system capable of detecting the genes of pathogens directly down to the sub-ng level without separate analysis equipment by applying the detection color value derived using the colorimetric sensor system according to the present disclosure to a smartphone application (app).